The coat protein complex II (COPII) mediates the sorting and export of cargo proteins from the endoplasmic reticulum (ER). We combine well-defined in vitro experimental systems that reconstitute COPII activities on ER membranes and liposomes, with cellular studies to provide a mechanistic view of the COPII-mediated ER export event. In aim 1 of the studies, we will examine the hypothesis that GTPase regulated dissociation of Sar1 from constricted membranes destabilizes vesicle necks to mediate COPII-vesicle fission. We will monitor the activity of purified COPII proteins on defined liposomes to examine the fission event. In aim 2 of the studies, we will examine the hypothesis that Sar1 assembly on the membrane regulates fission, and further explore the role of Sar1-induced membrane perturbation in the activation of lipid processing enzymes as a mechanism that couple lipid remodeling with vesicle formation. Structure-function studies on Sar1 will define the basis for the activity of Sar1 on membranes using liposomes and the information will be utilized in cellular models that recapitulate the synchronized export of different cargo proteins from the ER. In aim 3 we will define the molecular basis for lipid-binding specificities of COPII-interacting proteins, using defined liposomes and ER membranes. Cellular studies will complement the analysis to define the role of selective lipid recognition in supporting the nucleation of COPII at defined ER exit sites. Importantly, a large number of human diseases are derived from mistakes in protein sorting at the ER. Developing mechanistic understanding of the molecular basis for COPII functions in ER export should enable the development of future effective therapeutics